Protein kinases are a family of enzymes that catalyze the transfer of a phosphate group from ATP to the hydroxyl groups on tyrosine or serine/threonine residues of a client protein regulating several signal transduction pathways and crucial biological processes like cell proliferation, differentiation, survival, invasion and migration.
Up today, more than 500 members of the human protein kinase family have been discovered and classified into several subgroups. Certain human diseases, and more specifically cancers, are characterized by overexpression, amplification, mutations or rearrangements that induce aberrant activity of one or more protein kinases. The evident correlation between cancer development and dysregulation of protein kinases triggered the research of novel therapeutics for the modulation of biological activity of this class of proteins. First generation of approved protein kinase inhibitors provided potent drugs and successful clinical response during the treatment of some type of cancer. However, for a percentage of patients, therapeutic use of protein kinase inhibitors is very often associated to drug resistance leading to disease relapse. Drug resistance is mediated by several mechanisms such as secondary point mutations, activation of alternative survival and proliferation signaling pathways or gene amplification. Mutations of specific residues in the target kinase following a prolonged administration of an inhibitor represent the most relevant factors in acquiring drug resistance. Following kinase inhibitor treatment, amongst others, a frequently observed mutation is the one of the so called gatekeeper residue in the ATP binding site. For example within the protein kinase ALK, mutation from leucine to methionine of the gatekeeper residue 1196 confers drug resistance in about 30% of patients treated with Crizotinib, the drug approved by FDA as first line treatment of ALK positive non-small cell lung cancer. Development of drug resistance is thus a major limitation of currently available medications and the need for compounds useful to treat disease relapse is strongly emerging.
Amongst protein kinases heavily involved in human diseases, it is worth mentioning kinases such as ALK, RET, ROS1, DDR2, DDR1, PDGFR, EPHA2, EPHA3, EphB2, EphB4, ABL, KIT, FGFR2, FGFR3, VEFGR, IGF1R, p38α, JNK1, AXL, CDK2, CDKS, FLT3, MET, MER, MAP4K2, TYK2, RON, BRAF, TIE2, JAK1, JAK2, JAK3, IRAK4, IRAK1, EGFR, CSF1R, HCK, NIK, LCK, HER2, HER3, HER4, LYN, SRC, PKC6, RIPK2, BTK, CAMKK2, FGR, TRKA, TRKB and TRKC. These proteins show a leading role or cause oncogene addiction in several human cancers or in central nervous system and metabolic diseases like leukemia, breast cancer, prostate cancer, neuroblastoma, lung cancer, melanoma, thyroid cancer, medulloblastoma, pancreas cancer, lymphoma, Parkinson's disease, Alzheimer's disease.
Amongst mentioned protein kinases, the anaplastic lymphoma kinase ALK is regarded as a particularly relevant therapeutic target because is responsible for certain cancer pathologies. ALK is a transmembrane receptor tyrosine-kinase protein belonging to the superfamily of insulin receptor. The structure of ALK, the oncogenic activation and pharmacological inhibition are deeply discussed in Pharmacological Research (2013), 68(1): 68-94.
The ALK kinase is essentially expressed in the central and peripheral nervous system. ALK is constituted by a long extra-cellular domain (1020 amino-acid residues in humans), a 21 residues trans-membrane domain and a 561 residues intracellular tyrosine catalytic domain. ALK plays a relevant role during embryo development and its role decreases after birth. An aberrant activity of ALK kinase associated to specific gene translocations, point mutations, gene amplification and/or overexpression is clearly linked to cancer development such as non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), inflammatory myofibroblastic tumors (IMT), melanoma, glioblastoma, thyroid carcinoma, colorectal cancer and neuroblastoma.
The ALK catalytic domain was initially identified in ALCL cancer cells where the fusion protein NPM-ALK was observed. This fusion protein is the result of a translocation between the NPM1 and the ALK proteins. About 80% of all ALCL patients are characterized by the presence of the NPM-ALK fusion protein. Several other oncogenic fusion proteins involving ALK protein have been identified like TPM3-ALK in 12%-18% of inflammatory myofibroblastic tumors or the EML4-ALK in about 5% of NSCLC.
The amino terminal dimerization of ALK fusion proteins takes place in the cellular cytoplasm triggering the kinase domain activation which in turn causes the aberrant phosphorylation of intracellular substrates involved in proliferation (RAS/RAF/MEK/ERK) and survival (JAK/STAT, PI3/AKT). In addition, more than 20 activating point mutations have been identified in the pathogenesis of pediatric neuroblastoma associated to overexpression and/or amplification of ALK protein.
High level of mutated ALK protein and its fusion proteins in cancer tissues and low level in normal human tissues make the ALK protein an attractive target for the discovery of novel anti-cancer agents. A few ALK inhibitors have already been approved by FDA and many others are under clinical investigation. Amongst them Crizotinib is the first ALK inhibitor approved in 2011 by FDA for the treatment of ALK positive NSCLC patients. However, in about 30% of treated patients with Crizotinib, development of drug resistance has been observed. Most common cause of drug resistance is the mutation of the ALK gatekeeper residue L1196M that impairs the clinical efficacy of Crizotinib. Recent patents on ALK inhibitors are reported in Expert Opinion Therapeutic Patents, (2014) 24(4): 417-442).
In a certain percentage of NSCLC, genetic rearrangements involving the gene ROS1, which encodes the receptor tyrosine kinase ROS1 (ROS proto-oncogene 1 receptor tyrosine kinase), have been reported as well. The protein kinase ROS1, which belongs to the superfamily of insulin receptor, may function as a receptor for cell growth or differentiation factors. The approved ALK inhibitor Crizotinib targets in vitro also the ROS1 kinase. In clinical trials Crizotinib showed efficacy in patients ROS1 positive affected by NSCLC (J. Clin. Oncol. 2012, 30: 863-870; N. Engl. J. Med. 2014, 371: 1963-71). Like observed for ALK positive treated patients, also with kinase ROS1 resistance to Crizotinib therapy caused by mutations has been observed during clinical development. For example, mutations G2032R and L2026M have been detected in patients refractory to Crizotinib therapy. Protein kinase ROS1 is also expressed both in wild-type or mutated forms in several other tumors like glioblastoma, breast, liver, colon, kidney and stomach cancer. Moreover at least five oncogenic fusion proteins with the kinase ROS1 have been reported (Med. Res. Rev. 2011, 31(5): 794-818).
The rearranged during transfection proto-oncogene encodes the transmembrane receptor tyrosine kinase RET, a protein involved in several human cancers. Oncogenic mutations and rearrangements of RET kinase have been discovered in leukemia, thyroid, colon and lung cancer. RET fusion proteins in lung cancer have been described for the first time in 2012 making the RET kinase a relevant and novel target for a subpopulation of patients like the ALK and ROS1 protein kinases (Nat. Med. 2012, 18 (3), 382-384). Overexpression of RET kinase has been reported also in breast, pancreas and brain cancer. Resistance causing mutations have been described also for RET kinase with the frequent mutation of the gatekeeper residue V804M (Nat. Rev. Cancer, 2014, 14, 173-186).
Discoidin domain receptor 1 (DDR1) and discoidin domain receptor 2 (DDR2) are transmembrane protein receptor tyrosine kinases that play a crucial role in cancer development. DDR1 and DDR2 interact with collagens, important components of the extracellular matrix that directs crucial cellular processes like migration, motility, proliferation and differentiation. Dysregulation of DDR1 and DDR2 have been linked to diseases like fibrotic disorders, osteoarthritis and various types of cancers. Recently mutations of DDR2 protein kinase have been described as oncogenic drivers in about 4% of patients with lung squamous cell carcinoma (SCC). Mutations L239R and I638F are selectively sensitive to the non-selective DDR2 kinase inhibitor Dasatinib. Two patients with SCC carrying the mutation S768R exhibited a significant shrinkage of tumor size after treatment with Dasatinib (J. Med: Chem. 2015, (58), 3287-3301). Metastatic development is also strongly sustained by DDR1 and DDR2 protein kinases (Cancer Metastasis Rev. 2012, 31 (1-2), 295-321). In ductal breast carcinoma high expression of DDR2 has been described as the driver of metastatic process (Nat. Cell Biol. 2013, 15, 677-687). Acquired resistance to Dasatinib treatment has been observed in DDR2-dependent lung cancer cell lines where the mutation T654I of the gatekeeper residue has been described as responsible for drug resistance (Mol. Cancer Ther. 2014, 13, 475-482).
Erythropoietin-producing hepatocellular carcinoma receptor tyrosine kinases A and B (EphA, EphB receptors) are involved in various cellular signaling network with their ligands ephrins. EphA/EphB have been reported to be highly expressed in several type of cancer like melanoma, neuroblastoma, glioblastoma, prostate, lung, colon, thyroid, liver, breast and ovarian cancer (J. Cell. Mol. Med. 2012, 16 (12), 2894-2909). Particular interest is focused on protein kinase EphB2 that is the oncogenic driver of ependymoma, a rare type of brain and spinal cord cancer for which surgery or radiotherapy are the only available therapies due to inefficacy of standard chemotherapy in a large population of patients (Nature 2010, 466, 632-636). An aberrant activity of EphB2 has been observed in adhesion and cellular invasion in pediatric medulloblastoma (Neuro-Oncology 2012, 14(9): 1125-1135). EphB2, with other protein kinases like AXL, FGFR2, IGF1R and RET is activated in osteosarcoma cell lines supporting metastatic development. EphB2 is thus an attractive therapeutic target for treatment of osteosarcoma (Oncogenesis 2012, 1 (11), e34). Moreover mutations in protein kinases EphA3 and EphB2 have been identified on a panel of 52 pancreatic exocrine neoplasms (PLoS One 2010, 5(9), e12653).
The receptor tyrosine kinase family includes the members TRKA, TRKB and TRKC and plays an important role during the development and maintenance of the central and peripheral nervous system. In particular, TRKA and TRKB are involved in neuroblastoma Inhibition of TRK kinase proteins activity might be a useful therapy for the treatment of other type of cancers where these targets are overexpressed like in prostate, lung, colon, pancreas and breast cancer. For example TRKB protein kinase promotes the metastatic process of lung adenocarcinoma and is an important target for the treatment of metastatic lung cancer (Proc. Natl. Acad. Sci. USA 2014, 111 (28), 10299-10304).
Mutations in the NTRK1 gene, which encodes the TRKA kinase, for example are often present in cancers such as thyroid papillary carcinoma. Mutations occur through rearrangements of genetic material that combine the NTRK1 gene with another gene. These rearrangements give rise to mutant proteins known as oncoproteins that, unlike normal TRKA protein, do not require activation by binding to the protein NGFβ. The constant activation of the protein triggers signaling for cells growth and proliferation leading to thyroid papillary carcinoma. NTRK1 rearrangements were identified in human cell lines of colorectal cancer and observed even in patients with lung cancer. They lead to constitutive activation of the kinase activity and are oncogenic. In vitro treatment of cells expressing forms of rearranged NTRK1 with inhibitors of the TRKA kinase activity inhibits autophosphorylation of TRKA and cell growth (Nat. Med. 2013, 19 (11), 1469-1472). Finally, TRKA TRKB and TRKC protein kinases may be important biological mediators in pain-related disorders such as rheumatoid arthritis, Crohn's disease, neuropathic pain of traumatic origin, depression, fibromyalgia and irritable bowel syndrome.
RIPK2 kinase (Receptor interacting protein kinase-2), also known as RIP2 or RICK CARDIAK, is a serine-threonine protein kinase having also a tyrosine kinase activity (Genes & Dev. 2010, 24:2666-2677). Its C-terminal domain interacts with the cytoplasmic receptors NOD1 and NOD2 that play an important role in innate immune response. After activation, RIPK2 is associated with NOD1 and NOD2 and acts as a molecular scaffold to aggregate other kinases such as IKKα/β and TAK1/γ, involved in the activation of NF-κB and MAP kinases. The lack of regulation of its signal was related to auto-inflammatory diseases, in particular the development of Crohn's disease. In vivo pharmacological inhibition of RIPK2 leads to a marked improvement of the disease in a spontaneous model of ileitis due to Crohn's disease (J. Biol. Chem. 2014, 289, 29651-29664).
RIPK2 protein kinase is implicated in the migration and invasion of tumor cells. Recent findings show that the expression of the RIPK2 gene is significantly increased in triple-negative breast tumors (negative for estrogen receptors, progesterone and Her2/Neu-Her2) compared to other clinical subtypes and high RIPK2 gene expression correlates with a decrease in progression-free survival of the disease. These studies show that the RIPK2 gene is an independent prognostic marker that stimulates the processes of metastasis in patients with advanced breast cancer.
Finally the inhibition of the RIPK2 kinase increases the chemo sensitivity of cancer cells to docetaxel and decreases both the tumor and lung metastases in experimental models of mammary tumor (Breast Cancer Res. 2014, 16: R28). RIPK2 inhibitors containing the indazole nucleus structure are described in WO2011120025.
The non-receptor tyrosine kinase FYN is a protein that belongs to the SRC kinases family (SFKs) and is involved in normal physiological conditions in signal transduction pathways in the nervous system as well as in the development and activation of T lymphocytes. In cancer, FYN contributes to development and progression of melanoma, glioblastoma, squamous cell carcinoma and breast carcinoma. Recently FYN has been reported to play a key role in Tamoxifen-resistant ER positive breast cancer cell lines (Pharmacol. Res. 2015, 100, 250-254). Activating mutations of FYN have been also observed in integrated molecular analysis of adult T cell leukemia/lymphoma (ATL) (Nat. Genet. 2015, 47 (11), 1304-1315).
Alzheimer's disease is a neurodegenerative disease with a high incidence in the population over 85 years. The disease is characterized by the presence of plaques of beta-amyloid peptide (Aβ) and neurofibrillary tangles. There are numerous evidences involving the non-receptor tyrosine kinase Fyn in the pathogenesis of Alzheimer's disease. Fyn is involved in synaptic plasticity and plays a role in the regulation of Aβ production mediating synaptic deficits and Aβ-induced neurotoxicity. Fyn tyrosine phosphorylation induces tau protein activation that has been observed in neurofibrillary tangles in AD brain. Recent studies demonstrate that Aβ activates Fyn by binding to cellular prion protein on cell surface of Aβ oligomers. The interaction of Fyn with both Aβ and tau protein makes Fyn an important potential therapeutic target for the treatment of Alzheimer's disease (J. Alzheimer's Dis. 2011, 27(2), 243-252; Alzheimer's Research & Therapy, 2014, 6:8). Finally FYN regulates microglial neuroinflammatory responses in cell culture and animal models of Parkinson's disease making FYN a relevant and potential translational target for intervention of progressive neurodegenerative diseases (J. Neurosc. 2015, 35(27): 10058-10077).
The protein MAP4K2 or GCK kinase is a mitogen-activated protein kinase (MAPK) reported to be involved in pathogen associated molecular pattern signaling and systemic inflammation through JNK and p38 pathways. Moreover, in cancer disease MAP4K2 has been described to be a player upstream in the NFkβ pathway regulating the response of colorectal cancer to RAF inhibition. Recently GCK protein kinase has been also described as relevant target of mutant NRAS signaling especially in acute leukemia cell line with NRAS mutations (Blood 2015, 125 (20), 3133-3143).
The interleukin-1 receptor-associated kinases (IRAKs) are key mediators of toll-like receptor (TLR) and interleukin-1 receptor (IL1R) signaling processes. TLR/IL1R-mediated signaling controls diverse cellular processes including inflammation, apoptosis and cellular differentiation. IRAKs are categorized as serine/threonine protein kinases but only IRAK1 and IRAK4 display kinase activity. Human epidemiological studies as well as transgenic mouse models have linked genetic variations in IRAK genes to a collection of diverse diseases including inflammation and cancer. In particular, IRAK4 has been reported to be involved in melanoma, lymphoma, leukemia and breast cancer (Br. J. Cancer 2015, 112, 232-237).
The protein kinase JAK2 is a non-receptor tyrosine kinase that belongs to the Janus family of kinases (JAKs) in addition to JAK1 and JAK3 members. JAK2 is involved in several myeloproliferative diseases like essential thrombocythemia or primary myeolofibrosys. Mutational studies revealed that the somatic mutation V617F, within the pseudokinase domain of JAK2, is associated to disease development making the JAK2 protein kinase constitutively active. The prevalence of JAK2 activation in certain patients with myeloproliferative disorders provided a strong rationale for the exploration of new therapeutics to treat such diseases culminating with the discovery of Ruxolitinib, the first JAK1-JAK2 dual inhibitor approved by FDA. However myeloproliferative cells may develop an adaptive form of resistance to JAK2 inhibitors, termed persistence, causing the lack of pathologic remission despite clinical benefits for patients. Thus novel approaches providing inhibitors that could inactivate the JAK2 kinase activity more efficiently are urgently needed (Blood 2013, 122 (13), 2167-2175).